A multi-chamber implantable heart stimulation device is provided, adapted to inhibit anodal stimulation at a bipolar right chamber lead induced by a unipolar left chamber lead. The device includes a control unit adapted to provide to the unipolar left chamber lead a pace pulse having a trailing edge without a sharp cutoff. The control unit may be adapted to supply the pace pulse to the unipolar left chamber lead without a fast recharge period. The control unit may be adapted to supply to the unipolar left chamber lead a pace pulse having a trailing edge with a gradual decay, a ramp, steps, or a non-linear function. The control unit may be adapted to supply a different waveform to the bipolar right chamber lead than the pace pulse for the unipolar left chamber lead. For example, a conventional pacing pulse may be supplied to the bipolar right chamber lead.

Conventional Implantable Cardiac Devices (FIGS. 1 and 2)
FIG. 1 shows a conventional stimulation device 10 in electrical communication with a patient's heart 12 by way of three leads, 20, 24 and 30, suitable for delivering multi-chamber stimulation and shock therapy. To sense atrial cardiac signals and to provide right atrial chamber stimulation therapy, the stimulation device 10 is coupled to an implantable right atrial lead 20 having at least an atrial tip electrode 22, which typically is implanted in the patient's right atrial appendage, and an atrial ring electrode 23. To sense left atrial and ventricular cardiac signals and to provide left chamber pacing therapy, the stimulation device 10 is coupled to a "coronary sinus" lead 24 designed for placement in the "coronary sinus region" via the coronary sinus or for positioning a distal electrode adjacent to the left ventricle and/or additional electrode(s) adjacent to the left atrium. As used herein, the phrase "coronary sinus region" refers to the vasculature of the left ventricle, including any portion of the coronary sinus, great cardiac vein, left marginal vein, left posterior ventricular vein, middle cardiac vein, and/or small cardiac vein or any other cardiac vein accessible by the coronary sinus. Accordingly, an exemplary coronary sinus lead 24 is designed to receive atrial and ventricular cardiac signals and to deliver left ventricular pacing therapy using at least a left ventricular tip electrode 26, left atrial pacing therapy using at least a left atrial ring electrode 27, and shocking therapy using at least a left atrial coil electrode 28.
The stimulation device 10 is also shown in electrical communication with the patient's heart 12 by way of an implantable right ventricular lead 30 having, in this embodiment, a right ventricular tip electrode 32, a right ventricular ring electrode 34, a right ventricular (RV) coil electrode 36, and an SVC coil electrode 38. Typically, the right ventricular lead 30 is transvenously inserted into the heart 12 so as to place the right ventricular tip electrode 32 in the right ventricular apex so that the RV coil electrode will be positioned in the right ventricle and the SVC coil electrode 38 will be positioned in the superior vena cava. Accordingly, the right ventricular lead 30 is capable of receiving cardiac signals, and delivering stimulation in the form of pacing and shock therapy to the right ventricle.
FIG. 2 illustrates a simplified block diagram is shown of the multi-chamber implantable stimulation device 10, which is capable of treating both fast and slow arrhythmias with stimulation therapy, including cardioversion, defibrillation, and pacing stimulation. While a particular multi-chamber device is shown, this is for illustration purposes only, and one of skill in the art could readily duplicate, eliminate or disable the appropriate circuitry in any desired combination to provide a device capable of treating the appropriate chamber(s) with cardioversion, defibrillation and pacing stimulation.
The housing 40 for the stimulation device 10, shown schematically in FIG. 2, is often referred to as the "can", "case" or "case electrode" and may be programmably selected to act as the return electrode for all "unipolar" modes. The housing 40 may further be used as a return electrode alone or in combination with one or more of the coil electrodes, 28, 36 and 38, for shocking purposes. The housing 40 further includes a connector (not shown) having a plurality of terminals, 42, 43, 44, 46, 48, 52, 54, 56, and 58 (shown schematically and, for convenience, the names of the electrodes to which they are connected are shown next to the terminals). As such, to achieve right atrial sensing and pacing, the connector includes at least a right atrial tip terminal (AR TIP) 42 adapted for connection to the atrial tip electrode 22 and a right atrial ring (AR RING) electrode 43 adapted for connection to right atrial ring electrode 23. To achieve left chamber sensing, pacing and shocking, the connector includes at least a left ventricular tip terminal (VL TIP) 44, a left atrial ring terminal (AL RING) 46, and a left atrial shocking terminal (AL COIL) 48, which are adapted for connection to the left ventricular ring electrode 26, the left atrial tip electrode 27, and the left atrial coil electrode 28, respectively. To support right chamber sensing, pacing and shocking, the connector further includes a right ventricular tip terminal (VR TIP) 52, a right ventricular ring terminal (VR RING) 54, a right ventricular shocking terminal (RV COIL) 56, and an SVC shocking terminal (SVC COIL) 58, which are adapted for connection to the right ventricular tip electrode 32, right ventricular ring electrode 34, the RV coil electrode 36, and the SVC coil electrode 38, respectively.
At the core of the stimulation device 10 is a programmable microcontroller 60, which controls the various modes of stimulation therapy. As is well known in the art, the microcontroller 60 (also referred to herein as a control unit) typically includes a microprocessor, or equivalent control circuitry, designed specifically for controlling the delivery of stimulation therapy and may further include RAM or ROM memory, logic and timing circuitry, state machine circuitry, and I/O circuitry. Typically, the microcontroller 60 includes the ability to process or monitor input signals (data) as controlled by a program code stored in a designated block of memory. The details of the design and operation of the microcontroller 60 are not critical to the invention. Rather, any suitable microcontroller 60 may be used that carries out the functions described herein. The use of microprocessor-based control circuits for performing timing and data analysis functions are well known in the art.
As shown in FIG. 2, an atrial pulse generator 70 and a ventricular pulse generator 72 generate pacing stimulation pulses for delivery by the right atrial lead 20, the right ventricular lead 30, and/or the coronary sinus lead 24 via an electrode configuration switch 74. It is understood that in order to provide stimulation therapy in each of the four chambers of the heart, the atrial and ventricular pulse generators, 70 and 72, may include dedicated, independent pulse generators, multiplexed pulse generators, or shared pulse generators. The pulse generators, 70 and 72, are controlled by the microcontroller 60 via appropriate control signals, 76 and 78, respectively, to trigger or inhibit the stimulation pulses.
The microcontroller 60 further includes timing control circuitry 79 which is used to control the timing of such stimulation pulses (e.g., pacing rate, atrio-ventricular (AV) delay, atrial interconduction (A-A) delay, or ventricular interconduction (V-V) delay, etc.) as well as to keep track of the timing of refractory periods, blanking intervals, noise detection windows, evoked response windows, alert intervals, marker channel timing, etc., which is well known in the art. Switch 74 includes a plurality of switches for connecting the desired electrodes to the appropriate I/O circuits, thereby providing complete electrode programmability. Accordingly, the switch 74, in response to a control signal 80 from the microcontroller 60, determines the polarity of the stimulation pulses (e.g., unipolar, bipolar, combipolar, etc.) by selectively closing the appropriate combination of switches (not shown) as is known in the art.
Atrial sensing circuits 82 and ventricular sensing circuits 84 may also be selectively coupled to the right atrial lead 20, coronary sinus lead 24, and the right ventricular lead 30, through the switch 74 for detecting the presence of cardiac activity in each of the four chambers of the heart. Accordingly, the atrial (ATR. SENSE) and ventricular (VTR. SENSE) sensing circuits, 82 and 84, may include dedicated sense amplifiers, multiplexed amplifiers, or shared amplifiers. The switch 74 determines the "sensing polarity" of the cardiac signal by selectively closing the appropriate switches, as is also known in the art. In this way, the clinician may program the sensing polarity independent of the stimulation polarity. Each sensing circuit, 82 and 84, preferably employs one or more low power, precision amplifiers with programmable gain and/or automatic gain control, bandpass filtering, and a threshold detection circuit, as known in the art, to selectively sense the cardiac signal of interest. The automatic gain control enables the device 10 to deal effectively with the difficult problem of sensing the low amplitude signal characteristics of atrial or ventricular fibrillation. The outputs of the atrial and ventricular sensing circuits, 82 and 84, are connected to the microcontroller 60 which, in turn, are able to trigger or inhibit the atrial and ventricular pulse generators, 70 and 72, respectively, in a demand fashion in response to the absence or presence of cardiac activity in the appropriate chambers of the heart.
For arrhythmia detection, the device 10 utilizes the atrial and ventricular sensing circuits, 82 and 84, to sense cardiac signals to determine whether a rhythm is physiologic or pathologic. As used herein "sensing" is reserved for the noting of an electrical signal, and "detection" is the processing of these sensed signals and noting the presence of an arrhythmia. The timing intervals between sensed events (e.g., P-waves, R-waves, and depolarization signals associated with fibrillation which are sometimes referred to as "F-waves" or "Fib-waves") are then classified by the microcontroller 60 by comparing them to a predefined rate zone limit (i.e., bradycardia, normal, low rate VT, high rate VT, and fibrillation rate zones) and various other characteristics (e.g., sudden onset, stability, physiologic sensors, and morphology, etc.) in order to determine the type of remedial therapy that is needed (e.g., bradycardia pacing, antitachycardia pacing, cardioversion shocks or defibrillation shocks, collectively referred to as "tiered therapy"). Similar capabilities would exist on the atrial channel with respect to tachycardias occurring in the atrium. These would be atrial tachycardias (AT), more rapid atrial tachycardias (Atrial Flutter) and atrial fibrillation (AF).
Cardiac signals are also applied to the inputs of an analog-to-digital (A/D) data acquisition system 90. The data acquisition system 90 is configured to acquire intracardiac electrogram signals, convert the raw analog data into a digital signal, and store the digital signals for later processing and/or telemetric transmission to an external device 102. The data acquisition system 90 is coupled to the right atrial lead 20, the coronary sinus lead 24, and the right ventricular lead 30 through the switch 74 to sample cardiac signals across any pair of desired electrodes.
The microcontroller may include a capture-based tachycardia detection unit, which operates to detect a tachycardia based on loss of capture of pacing pulses. In the primary example described herein, the tachycardia detection unit operates to detect AF based on loss of capture of atrial pacing signals during preventive overdrive pacing in the atrium. Accordingly, the capture-based tachycardia detection unit is used in conjunction with a preventive overdrive pacing unit for controlling overdrive pacing of the heart. One particularly effective overdrive pacing technique, referred to herein as dynamic atrial overdrive (DAO) pacing, is described in U.S. Pat. No. 6,519,493 to Florio et al., entitled METHODS AND APPARATUS FOR OVERDRIVE PACING HEART TISSUE USING AN IMPLANTABLE CARDIAC STIMULATION DEVICE, which is incorporated by reference herein. With DAO, the overdrive rate is controlled to remain generally uniform and, in the absence of a tachycardia, is adjusted upwardly or downwardly only occasionally. Dynamic overdrive techniques are also applicable to the ventricles. Exemplary dynamic ventricular overdrive (DVO) techniques are described in U.S. patent applications: 1) Ser. No. 10/456,060 to Park et al., entitled SYSTEM AND METHOD FOR DYNAMIC VENTRICULAR OVERDRIVE PACING, filed Jun. 6, 2003 Ser. No. 10/456,058, entitled SYSTEM AND METHOD FOR DYNAMIC VENTRICULAR OVERDRIVE PACING, Jun. 6, 2003, which applications are also incorporated herein by reference. In one example, the overdrive pacing unit operates continuously in the absence of a tachycardia so as to reduce the likelihood of the onset of a tachycardia. In other examples, preventive overdrive pacing is suspended while the patient is asleep. It is also suspended when the patient is in a tachycardia that has resulted in the enabling of the Automatic Mode Switch algorithm. In any case, if a tachycardia is detected during preventive overdrive pacing by the tachycardia detection unit, an ATP unit is activated to deliver antitachycardia pacing to the heart in an effort to terminate the tachycardia.
The ATP unit may administer ATP in accordance with any of a variety of ATP techniques. Exemplary patents describing ATP techniques include U.S. Pat. No. 6,101,414, to Mark Kroll, entitled METHOD AND APPARATUS FOR ANTITACHYCARDIA PACING WITH AN OPTIMAL COUPLING INTERVAL, and U.S. Pat. No. 5,431,689 to Weinberg et al., entitled IMPLANTABLE STIMULATION SYSTEM AND METHOD FOR TERMINATING CARDIAC ARRHYTHMIAS, which are both incorporated by reference herein. Tachycardia may be detected during preventive overdrive pacing based, for example, upon detection of a true loss of capture of a preventive overdrive pacing pulse or upon detection of a loss of capture of a backup pulse subsequent to a PAC.
To detect loss of capture, the microcontroller also includes an automatic capture detection unit for detecting an evoked response from the heart in response to an applied stimulus. The capture detection unit verifies capture of both primary pacing pulses and any subsequent backup pulses. Capture occurs when an electrical stimulus applied to the heart is of sufficient energy to depolarize the cardiac tissue, thereby causing the heart muscle to contract. The capture detection unit detects a depolarization signal during a window following a stimulation pulse, the presence of which indicates that capture has occurred. Capture detection is performed on a beat-by-beat basis. If a primary pulse is not captured, a backup pulse unit delivers a backup pulse at a maximum pulse magnitude. The capture detection unit also detects whether the backup pulse captures.
Also included is a stimulation threshold search unit for automatically determining the current capture threshold of the patient, i.e. the minimum output sufficient to evoke capture, so that the output or pulse magnitude can be reset properly. This is commonly reported in terms of pulse amplitude as this is one of the programmable output parameters. While preventive overdrive pacing is performed, a stimulation search is automatically performed in circumstances wherein a primary pacing pulse is not captured but the backup pulse is captured. (If both the overdrive pulse and the backup pulse are not captured, ATP is instead activated.) Also, preferably, a capture threshold search is performed periodically to update the capture threshold regardless of whether any loss of capture is detected. Such capture threshold searches are preferably performed every eight hours. Typically, a capture threshold search begins at a desired starting point (either a high energy level or the level at which capture is currently occurring) and decreases the energy level until capture is lost. It then increments the output in 0.125 Volt steps until capture is restored. The value at which capture is restored is known as the capture threshold. Thereafter, a working margin or a safety margin is added to the capture threshold to yield a new pulse magnitude. A safety margin is a fixed multiple of the measured threshold. A working margin is a fixed value, e.g. 0.25 Volts above the measured threshold. In one implementation, the safety margin is provided by the high output backup pulse. The delivered output associated with the primary pulse is simply a working margin above the measured capture threshold.
Various techniques for implementing capture verification of atrial pacing pulses (i.e. atrial AutoCapture) are set forth in U.S. Pat. Nos. 6,434,428 to Sloman et al.; 6,311,089 to Mann et al.; 6,285,908 to Mann et al.; 6,263,244 to Mann et al.; 6,259,950 to Mann et al.; 6,243,606 to Mann et al.; and 6,101,416 to Sloman, which are incorporated herein by reference. Capture verification of ventricular pulses is described in 6,456,882 to Schloss; 6,456,881 to Bornzin et al.; and 6,345,201 to Sloman, et al, which are also incorporated herein by reference. See also U.S. Pat. No. 4,686,988 (Sholder); U.S. Pat. No. 4,969,467 (Callaghan et al.); and U.S. Pat. No. 5,350,410 (Kleks et al.), which patents are hereby incorporated herein by reference. A technique for implementing automatic capture verification during overdrive pacing is described in U.S. patent application Ser. No. 10/138,438, filed May 2, 2002, of Bradley et al., entitled METHOD AND APPARATUS FOR PROVIDING ATRIAL AUTOCAPTURE IN A DYNAMIC ATRIAL OVERDRIVE PACING SYSTEM FOR USE IN AN IMPLANTABLE CARDIAC STIMULATION DEVICE, which is incorporated herein by reference.
The microcontroller also includes a PAC detection unit 109 and a PAC response unit. The PAC detection unit detects PACs and the PAC response unit provides a pacing protocol for responding to the PAC. An exemplary PAC response protocol is described in U.S. Pat. No. 5,978,709 to Begemann et al., entitled PACEMAKER SYSTEM WITH IMPROVED TECHNIQUES FOR PREVENTING AND SUPPRESSING ATRIAL ARRHYTHMIAS, which is incorporated herein by reference.
Although shown as being components of the microcontroller, any or all of capture-based tachycardia detection unit, overdrive pacing unit, ATP unit, capture detection unit, stimulation threshold search unit, PAC detection unit, PAC response unit, and backup pulse unit could be instead implemented as separate components. Also, depending up on the particular component and the particular implementation, individual components may be configured to apply to the ventricles, the atria, or in some cases both.
The microcontroller 60 is further coupled to a memory 94 by a suitable data/address bus 96, wherein the programmable operating parameters used by the microcontroller 60 are stored and modified, as required, in order to customize the operation of the stimulation device 10 to suit the needs of a particular patient. Such operating parameters define, for example, pacing pulse amplitude, pulse duration, electrode polarity, rate, sensitivity, automatic features, arrhythmia detection criteria, and the amplitude, waveshape and vector of each shocking pulse to be delivered to the patient's heart 12 within each respective tier of therapy. Other pacing parameters include base rate, rest rate and circadian base rate.
Advantageously, the operating parameters of the implantable device 10 may be non-invasively programmed into the memory 94 through a telemetry circuit 100 in telemetric communication with the external device 102, such as a programmer, transtelephonic transceiver, or a diagnostic system analyzer. The telemetry circuit 100 is activated by the microcontroller by a control signal 106. The telemetry circuit 100 advantageously allows intracardiac electrograms and status information relating to the operation of the device 10 (as contained in the microcontroller 60 or memory 94) to be sent to the external device 102 through an established communication link 104. In the one embodiment, the stimulation device 10 further includes a physiologic sensor 108, commonly referred to as a "rate-responsive" sensor because it is typically used to adjust pacing stimulation rate according to the exercise state of the patient. However, the physiological sensor 108 may further be used to detect changes in cardiac output, changes in the physiological condition of the heart, or diurnal changes in activity (e.g., detecting sleep and wake states). Accordingly, the microcontroller 60 responds by adjusting the various pacing parameters (such as rate, AV Delay, V-V Delay, etc.) at which the atrial and ventricular pulse generators, 70 and 72, generate stimulation pulses. (V-V delay is typically used in only connection with independently programmable RV and LV leads for biventricular pacing.) While shown as being included within the stimulation device 10, it is to be understood that the physiologic sensor 108 may also be external to the stimulation device 10, yet still be implanted within or carried by the patient. A common type of rate responsive sensor is an activity sensor, such as an accelerometer or a piezoelectric crystal, which is mounted within the housing 40 of the stimulation device 10. Other types of physiologic sensors are also known, for example, sensors that sense the oxygen content of blood, respiration rate and/or minute ventilation, pH of blood, ventricular gradient, etc. However, any sensor may be used which is capable of sensing a physiological parameter that corresponds to the exercise state of the patient.
The stimulation device additionally includes a battery 110, which provides operating power to all of the circuits shown in FIG. 2. For the stimulation device 10, which employs shocking therapy, the battery 110 must be capable of operating at low current drains for long periods of time, and then be capable of providing high-current pulses (for capacitor charging) when the patient requires a shock pulse. The battery 110 must also have a predictable discharge characteristic so that elective replacement time can be detected. Accordingly, the device 10 preferably employs lithium/silver vanadium oxide batteries, as is true for most (if not all) current devices. As further shown in FIG. 2, the device 10 is shown as having an impedance measuring circuit 112 which is enabled by the microcontroller 60 via a control signal 114.
In the case where the stimulation device 10 is intended to operate as an implantable cardioverter/defibrillator (ICD) device, it must detect the occurrence of an arrhythmia, and automatically apply an appropriate antitachycardia pacing therapy or electrical shock therapy to the heart aimed at terminating the detected arrhythmia. To this end, the microcontroller 60 further controls a shocking circuit by way of a control signal. The shocking circuit generates shocking pulses of low (up to 0.5 joules), moderate (0.5-10 joules), or high energy (11 to 40 joules), as controlled by the microcontroller 60. Such shocking pulses are applied to the patient's heart 12 through at least two shocking electrodes, and as shown in this embodiment, selected from the left atrial coil electrode 28, the RV coil electrode 36, and/or the SVC coil electrode 38. As noted above, the housing 40 may act as an active electrode in combination with the RV electrode 36, or as part of a split electrical vector using the SVC coil electrode 38 or the left atrial coil electrode 28 (i.e., using the RV electrode as a common electrode).
Cardioversion shocks are of relatively low to moderate energy level (so as to minimize the current drain on the battery) and are usually between 5 to 20 joules. Typically, cardioversion shocks are synchronized with an R-wave. Defibrillation shocks are generally of moderate to high energy level (i.e., corresponding to thresholds in the range of 15 to 40 joules), delivered asynchronously (since R-waves may be too disorganized), and pertaining exclusively to the treatment of fibrillation. Accordingly, the microcontroller 60 is capable of controlling the synchronous or asynchronous delivery of the shocking pulses.
Congestive Heart Failure
Clinical evidence is revealing that patients suffering from cardiac diseases which affect the contractility of the heart muscle tissue rather than the conduction pathways, generally known as congestive heart failure or CHF, can benefit from cardiac pacing. CHF is a condition in which a weakened heart cannot pump enough blood to body organs. Heart failure may affect either the right side, left side, or both sides of the heart. As pumping action is lost, blood may back up into other areas of the body, including the liver, gastrointestinal tract, and extremities (right-sided heart failure), or the lungs (left-sided heart failure). Structural or functional causes of heart failure include high blood pressure (hypertension), valvular heart disease, congenital heart diseases, cardiomyopathy, heart tumor, and other heart diseases. Precipitating factors include infections with high fever or complicated infections, use of negative inotropic drugs (such as beta-blockers and calcium channel blocker), anemia, irregular heartbeats (arrhythmias), hyperthyroidism, and kidney disease.
Treatment typically involves pacing on both sides of the heart. In such patients, pacing in the atria and ventricles effectively resynchronizes heart chamber contractions thereby improving hemodynamic function of the heart. Biventricular pacing has proven to be an effective therapy or treating patient with congestive heart failure.
A system and method for monitoring progression of cardiac disease state using physiologic sensors is disclosed in U.S. Pat. No. 6,572,557, by Tchou, et al., issued Jun. 3, 2003, which describes a technique for monitoring physiological parameters associated with the progression, stabilization, or regression of symptoms of heart disease such as congestive heart failure (CHF), which is herein incorporated by reference in its entirety. A system and method for evaluating risk of mortality due to congestive heart failure using physiologic sensors is disclosed in U.S. Pat. No. 6,645,153 by Kroll et al., issued Nov. 11, 2003, which is herein incorporated by reference in its entirety. One method and apparatus for biventricular stimulation and capture monitoring is disclosed in published U.S. Patent application publication number 20010049543, filed May 1, 2001, by Mark Kroll, herein incorporated by reference in its entirety.
For CHF the left side of the heart, as well as the right is paced. Unfortunately, when fitting a patient with an implantable pacing device, it can be difficult to pass a left-side lead into the coronary sinus vein, or the smaller final destination veins. To facilitate left side lead 24 implantation, the left side lead 24 may be unipolar, with a tip 26, but without the ring 27 shown in FIG. 1. The case 10 could be used as the return, but it sometimes causes stimulation of the muscle surrounding the case, which is uncomfortable to the patient. As an alternative, the ring 34 of the right ventricle lead 30 is used as the return path. Not only does this allow high current density around the tip 26 in the left ventricle for pacing, it also causes high current density around the ring 34. If the ring 34 is in contact with the heart muscle, it will stimulate the right ventricle along with the left ventricle. This is sometimes referred to as anodal stimulation.
Anodal stimulation causes the right ventricle to pace at the same time as the left ventricle. Ideally, however, the right side should be stimulated 20-40 milliseconds after the left side. If both sides are stimulated at the same time rather than 20-40 milliseconds apart, the patient can feel faint as the heart is not pumping as much blood.
Accordingly, what is needed is device that allows bi-ventricular pacing with a unipolar left ventricle lead without causing anodal stimulation in the right ventricle. Moreover, what is needed is a means to inhibit anodal stimulation in patients being treated with bi-chamber pacing for congestive heart failure.

Unipolar and bipolar lead cardiac pacemaker and method for inhibiting anode stimulation


An improved RF beacon for use in space/earth communications and positioning determination provides better spectral purity and frequency stability. A power supply provides regulated voltage and control signals to the beacon. Each of two oscillators operate at a slightly different frequency very near the aforementioned 16.7 MHz. The slight difference provides for a frequency selection with respect to the transmitter frequency spectra. The oscillator provides a signal to a plurality of multiplier circuits. The resulting multiplied frequency signals are fed to respective amplifiers. Some of the features of the radio frequency beacon include redundant switchable oscillators, a 1067 MHz signal having data modulation capabilities, the ability to change power levels, improved frequency stability and temperature control characteristics, and a triplexer to optionally combine the three outputs into a single port.

The present invention relates generally to radio frequency beacons (RF beacons) for use in space and on the ground (terrestrial), and more specifically to a radio frequency beacon capable of broadcasting multiple signals of different frequencies simultaneously, wherein one or more of the broadcasted signals can be phase modulated to include data in the signal.
One use for radio frequency beacons is to track the position of spacecraft that are in orbit outside the earth""s atmosphere. It is important to know the position of such spacecraft as exactly as possible in order to aim ground based systems that are in communication with the spacecraft. Position information is also important to accurately interpret scientific data collected by spacecraft instrumentation for transmission back to earth.
RF beacons deployed in space face tougher performance obstacles as compared to ground based RF beacons. Chiefly, an RF beacon in space must broadcast great distances through the earth""s atmosphere without a significant degradation of its signal purity. Furthermore, space based systems also face restrictions on size, weight, power, and quality or ruggedness of their design.
The present invention provides an improved RF beacon having better spectral purity and frequency stability over the current state of the art, in addition to more features. The radio frequency beacon of the present invention outputs fixed tones so that the position of the spacecraft in which it resides can be determined by means of Doppler tracking. Particular characteristics of the present invention include broadcasting coherent signals at 150 MHz and 400 MHz simultaneously thereby enabling the removal of ionospheric effects. A 1067 MHz signal can also be simultaneously broadcast. The 1067 MHz signal can be modulated with a data bit stream encoded by, or mixed with a xe2x80x9cgold codexe2x80x9d which is a pseudo-random code modulation scheme that allows telemetry data to be placed on the carrier without disturbing the overall phase of the 1067 MHz signal.
A power supply converts unregulated spacecraft bus power to the positive and negative DC voltages needed by the beacon electronics. There are two oscillators operating at approximately 16.7 MHz. Each oscillator operates at a slightly different frequency to provide for frequency selection with respect to the transmitter frequency spectra. The oscillators are redundant meaning that only one need be operating at any given time and the other serves as a backup in case of a failure, or if a slight frequency change is desired. The RF beacon can switch between the oscillators by remote command. Each oscillator also contains a heater control element to help maintain optimal performance. Whichever oscillator is in use simultaneously provides a signal to a times-8 and a times-9 multiplier circuit. The times-9 multiplied 16.7 MHz signal is approximately equivalent to a 150 MHz signal, which is then fed to an amplifier that provides a 150 MHz output signal. The times-8 multiplied 16.7 MHz signal is approximately equivalent to a 133 MHz signal. It is multiplied again by a times-3 multiplier to produce approximately a 400 MHz signal which is then fed to an amplifier that provides a 400 MHz output signal. A 1067 MHz output signal is obtained by multiplying the original 16.7 MHz oscillator reference signal by a times-8 multiplier and then by another times-8 multiplier. The resulting twice multiplied signal is fed to a phase modulator and is modulated by a pseudo random encoded data stream. The output of the phase modulator is approximately a 1067 MHz signal that is phase modulated. This phase modulated signal is then fed to an amplifier which outputs a 1067 MHz signal. All of the amplified output signals are then fed to individual antennas and broadcast. Another variant of the present invention combines the three outputs into a single port by means of a triplexer circuit. A single multi-band antenna can be used in this case to simplify spacecraft cabling and minimize the space needed for mounting antennas.
Some of the features of the RF beacon of the present invention include redundant switchable oscillators, a 1067 MHz signal having data modulation capabilities, the ability to change power levels, improved frequency stability and temperature control characteristics, and an optional three-way combined output with a single output port.
Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.


LERK-5 polypeptides are disclosed, along with DNA sequences, vectors and transformed host cells useful in producing LERK-5. The LERK-5 polypeptides bind to elk and to hek, which are members of the eph/elk family of receptor tyrosine kinases.

Proteins known as the receptor tyrosine kinases have an intrinsic kinase activity that is activated upon ligand binding. This class of proteins is characterized by conserved structural motifs within the catalytic domains (Hanks et al., Science, 242:42, 1988) and can be subdivided into families based on structural features of the regions N-terminal to the catalytic domain.
Boyd et al. (J. Biol. Chem., 267:3262, 1992) purified a cell surface glycoprotein exhibiting tyrosine kinase activity. The N-terminal amino acid sequence identified this protein as a member of the eph/elk family, and the protein was thus designated hek (human eph/elk-like kinase). A monoclonal antibody immunoreactive with hek was used to study hek expression on a number of human cell types (Boyd et al., supra). Hek antigen was detected on the human pre-B cell leukemia cell line LK63 (the cell line employed as the immunogen against which the antibody was raised) and the human T-cell leukemia cell line JM. The Raji B lymphoma cell line showed weak hek antigen expression, and the remaining cell lines tested (both normal and tumor cell lines, among which were hemopoietic cell lines that included pre-B and T-cell lines) were consistently negative. Of the normal and tumor tissue biopsy specimens that were also tested for hek antigen expression, none of the normal tissues was positive and only a very low proportion of hemopoietic tumors was positive.
Expression of hek transcripts in the above-described LK63 and JM cell lines, as well as the human T-cell leukemia cell line HSB-2, has been demonstrated by northern blot analysis (Wicks et al., Proc. Natl. Acad. Sci. USA, 89:1611, 1992). Nucleotide and amino acid sequences for an isolated hek cDNA clone are presented in Wicks et al., supra.
The cell surface protein designated elk is another member of the tyrosine kinase receptor family of proteins. A partial clone of elk was first discovered in a rat brain cDNA expression library that was screened for proteins expressing tyrosine kinase activity (Letwin et al., Oncogene 3:621, 1988). Later, a composite sequence spanning the entire elk coding region was derived from partial clones isolated from a rat brain cDNA library and a rat cerebellar brain library using the partial clone as a probe (Lhotak et al., Mol. Cell. Biol. 11:2496, 1991).
The hek and elk proteins are closely related to a number of other receptor tyrosine kinases, including the hek homologs mek4 and cek4 (Sajjadi et al. New Biol. 3:769, 1991); eek (Chan et al. Oncogene 6:1057, 1991); erk (Chan et al. supra.), eck (Lindberg et al. Mol. Cell. Biol. 10:6316, 1990); cek5 (Pasquale, E. B. Cell Regulation 2:523, 1991); and eph (Hirai et al. Science 238:1717, 1987). The proteins of this subfamily are related not only in their cytoplasmic domains, but also in their extracellular domains, which are 41 to 68% identical. Interestingly, the tissue distributions of these various receptors are diverse. For example, expression of elk mRNA has been reported to be limited to testis and brain (Lhotak et al., supra), whereas eck is found not only in these same two tissues but in lung, intestine, kidney, spleen, ovary, and skin as well.
Those ligands that have been identified for the receptor tyrosine kinases are a diverse group of proteins that affect the growth, differentiation, and survival of cells expressing the receptors. Ligands for hek and elk have been isolated, as discussed in more detail below.
Identification of any additional ligands for hek and elk that may exist would prove useful in investigating the nature of cellular processes regulated by signaling through these receptors. If enhancement or inhibition of a particular biological signal mediated through these receptors is desired, it is advantageous to identify each of the proteins that may play a role in transduction of such signals. Further, proteins that bind to certain receptors without initiating signal transduction are known, including interleukin-l receptor antagonist protein (Eisenberg et al., Nature 343:341, 1990; Hannum et al., Nature 343:336, 1990; and Carter et al., Nature 344:633, 1990). Identification of any additional proteins that bind hek or elk is desirable in order to determine whether such proteins function as antagonists.
The present invention provides a novel cytokine designated LERK-5 that binds to elk and to hek, which are members of the eph/elk family of receptor tyrosine kinases. The present invention also provides isolated DNA encoding the LERK-5 protein and expression vectors comprising the isolated DNA. A method for producing LERK-5 comprises cultivating host cells containing the expression vectors under conditions appropriate for expression of LERK-5 protein, and recovering the LERK-5. Antibodies directed against the LERK-5 protein are also disclosed.
A cDNA encoding a novel protein designated LERK-5, which binds to the cell surface receptors known as elk and hek, has been isolated in accordance with the present invention. Also provided are expression vectors comprising LERK-5 DNA. Methods for producing recombinant LERK-5 polypeptides involve cultivating host cells transformed with the expression vectors under conditions appropriate for expression of LERK-5, and recovering the expressed LERK-5. Purified LERK-5 protein is also encompassed by the present invention, including soluble forms of the protein comprising the extracellular domain.
The present invention also provides LERK-5 or fragments thereof that can act as immunogens to generate antibodies reactive therewith. In one embodiment, the antibodies are monoclonal antibodies specific for LERK-5.
Human LERK-5 cDNA was successfully isolated as described in example 1. The DNA sequence of the coding region of a human LERK-5 cDNA clone, and the amino acid sequence encoded thereby, are set forth in SEQ ID NO:1 and SEQ ID NO:2. The encoded protein comprises an N-terminal signal peptide (amino acids -25 to -1 of SEQ ID NO:2), an extracellular domain (amino acids 1 to 199), a transmembrane region (amino acids 200 to 225), and a cytoplasmic domain (amino acids 226 to 308). Binding of LERK-5 to the two cell surface receptors known as elk and hek is demonstrated in example 4.
A cell lysate containing a recombinant phage xcexgt10 vector comprising LERK-5 cDNA was deposited with the American Type Culture Collection on Jun. 16, 1994, and assigned accession no. ATCC 75815. The deposit was made under the terms of the Budapest Treaty. The recombinant xcexgt10 vector was that isolated in example I and identified as clone xcex6.
The novel cytokine disclosed herein is a ligand for elk, a rat cell surface receptor that is a member of the eph/elk family of receptor tyrosine kinases. Expression of elk mRNA has been detected in the brain and testis of rats (Lhotok et al., supra), and the possibility that elk is capable of oncogenic activation has been suggested (Letwin et al., supra). The cytokine additionally is a ligand for hek, which is another member of the eph/elk family of receptor tyrosine kinases (Boyd et al., J. Biol. Chem., 267:3262, 1992; Wicks et al., PNAS USA, 89:1611, 1992). Among the cell types on which hek is expressed are certain human leukemia cell lines.
The term xe2x80x9cLERK-5xe2x80x9d as used herein refers to a genus of polypeptides which are capable of binding elk and hek, and exhibit substantial homology to the LERK-5 amino acid sequences disclosed herein. Human LERK-5 is within the scope of the present invention, as are LERK-5 proteins derived from other mammalian species, including but not limited to murine, rat, bovine, porcine, or various primate species. As used herein, the term xe2x80x9cLERK-5xe2x80x9d includes membrane-bound proteins (comprising an extracellular domain, a transmembrane region, and a cytoplasmic domain) as well as truncated proteins that retain the elk-binding or hek-binding property. Such truncated proteins include, for example, soluble LERK-5 comprising only the extracellular (receptor binding) domain.
Other proteins that bind elk and hek have been identified. The LERK-5 of the present invention is a novel protein, distinct from these other elk-binding and hek-binding proteins.
One elk ligand is described in PCT application WO 94/11384. The nucleotide sequence of cloned cDNA (designated clone tele7) that encodes this elk ligand, as well as the amino acid sequence encoded thereby, are presented in WO 94/11384. The protein is a type 1 transmembrane protein comprising 346 amino acids, including an N-terminal signal peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.
Two hek ligand proteins (encoded by clones designated A2 and C6), which are 38% identical at the amino acid level, are described in U.S. Pat. No. 5,516,658. DNA and encoded amino acid sequences for cloned cDNA encoding the two hek ligand proteins are presented in U.S. Pat. No. 5,516,658. The encoded proteins each contain an N-terminal signal peptide, an extracellular domain, and a C-terminal hydrophobic region that contains signals for glycosyl-phosphatidylinositol (GPI) anchoring. After post-translational processing, both proteins are anchored to the cell surface via GPI linkage.
Another protein that has been found to bind elk and hek is designated B61. Nucleotide and encoded amino acid sequences for B61 cDNA are presented in Holzman et al. (Mol. Cell. Biol. 10:5830, 1990). B61 subsequently was found to bind elk and hek, as demonstrated in the binding assays described in U.S. Pat. No. 5,516,658.
Of these four proteins (B61 and the proteins encoded by clones tele7, A2, and C6), the LERK-5 of the present invention is most closely related (structurally) to the elk ligand encoded by tele7 (designated elk-L tele7 hereinafter). The amino acid sequences of the full length LERK-5 and elk-L tele7 proteins are 58.5% identical, and the DNA sequences are 61.4% identical. The sequences comprising the signal peptide and extracellular domain of elk-L and LERK-5 are 51.6% identical at the amino acid level, and 53.6% identical at the DNA level. The cytoplasmic domains are 74.7% identical at the amino acid level, and 75.5% identical at the DNA level.
The nucleotide sequence of a human expressed sequence tag (EST) for chromosome 13 is found in GenBank(copyright) under accession no. L13819. The source of the EST is identified as cDNA derived from mRNA from the brain of a 3-month old human female. When the 337 bp sequence disclosed for the EST is aligned with the elk-L tele7 nucleotide sequence, the sequence tag is 59.3% identical to the corresponding region of the elk-L tele7 DNA. In view of this degree of sequence homology, the present inventors sought to determine whether or not the sequence tag was part of a gene that encoded a biologically active protein, specifically, a protein that would bind elk or hek.
The GenBank record does not disclose any polypeptide encoded by the EST (e.g., does not indicate what the reading frame, if any, might be), much less suggest that any such polypeptide would bind elk or hek. Even if the sequence tag DNA were expressed in the reading frame elucidated by the cloning of LERK-5 DNA reported herein, the encoded amino acid sequence would lack three of the four cysteine residues that are conserved in the extracellular domains of the four above-described proteins that bind elk and hek. In the LERK-5 protein of the present invention, these cysteines are found at amino acids 37, 64, 76, and 128 of SEQ ID NO:2. A translate of the sequence tag would correspond to amino acids 79 to 190 of SEQ ID NO:2. Thus, the 337 bp sequence tag is not believed to encode a polypeptide capable of binding elk or hek.
The human LERK-5 cDNA isolated in example 1 below may be radiolabeled and used as a probe to isolate other mammalian LERK-5 cDNAs by cross-species hybridization. RNAs isolated from various cell lines or tissues derived from different mammalian species can be screened by Northern hybridization to identify a suitable source of mRNA for use in cloning a LERK-5 gene.
Fragments of the LERK-5 protein of SEQ ID NO:2 that are capable of binding elk or hek are encompassed by the present invention. One embodiment of the present invention provides soluble LERK-5 polypeptides. Soluble LERK-5 polypeptides comprise all or part of the extracellular domain of a native LERK-5 but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane. Soluble LERK-5 polypeptides advantageously comprise the native (or a heterologous) signal peptide when initially synthesized to promote secretion, but the signal peptide is cleaved upon secretion of LERK-5 from the cell. The soluble LERK-5 polypeptides that may be employed retain the ability to bind elk or hek. Soluble LERK-5 may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble LERK-5 protein is capable of being secreted.
Soluble LERK-5 may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired protein from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired protein. The presence of LERK-5 in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the desired protein. Soluble LERK-5 may be a naturally-occurring form of this protein.
The use of soluble forms of LERK-5 is advantageous for certain applications. Purification of the proteins from recombinant host cells is facilitated, since the soluble proteins are secreted from the cells. Further, soluble proteins are generally more suitable for intravenous administration.
Examples of soluble LERK-5 polypeptides include those comprising the entire extracellular domain of a native LERK-5 protein. One such soluble LERK-5 protein comprises amino acids 1 through 199 of SEQ ID NO:2. When initially expressed within a host cell, the soluble protein may additionally comprise one of the heterologous signal peptides described below that is functional within the host cells employed. Alternatively, the protein may comprise the native signal peptide, such that the LERK-5 comprises amino acids 25 through 199 of SEQ ID NO:2. DNA sequences encoding soluble LERK-5 proteins are encompassed by the present invention.
Truncated LERK-5, including soluble polypeptides, may be prepared by any of a number of conventional techniques. A desired DNA sequence may be chemically synthesized using known techniques. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. Oligonucleotides that reconstruct the 5xe2x80x2 or 3xe2x80x2 end of a DNA fragment to a desired point may be synthesized. The oligonucleotide may contain a restriction endonuclease cleavage site upstream of the desired coding sequence and position an initiation codon (ATG) at the 5xe2x80x2-terminus of the coding sequence. Linkers containing restriction endonuclease cleavage site(s) may be employed to insert the desired DNA fragment into an expression vector. The well known polymerase chain reaction procedure also may be employed to isolate a DNA sequence encoding a desired protein fragment. Oligonucleotides that define the termini of the desired fragment are employed as primers in the PCR. As a further alternative, known mutagenesis techniques may be employed to insert a stop codon at a desired point, e.g., immediately downstream of the codon for the last amino acid of the extracellular domain.
Regarding the foregoing discussion of signal peptides and the various domains of the LERK-5 protein, the skilled artisan will recognize that the above-described boundaries of such regions of the protein are approximate. For example, although computer programs that predict the site of cleavage of a signal peptide are available, cleavage can occur at sites other than those predicted. Further, it is recognized that a protein preparation can comprise a mixture of protein molecules having different N-terminal amino acids, due to cleavage of the signal peptide at more than one site. In addition, the exact boundaries of a transmembrane region may differ from those predicted by a computer program. Post-translational processing, which can vary according to the particular expression system employed, may yield proteins having N- or C-terminal amino acids that differ from those described above. Such variants that retain the desired biological activity are included among the LERK-5 polypeptides of the present invention.
The present invention provides purified LERK-5 polypeptides, both recombinant and non-recombinant. Variants and derivatives of native LERK-5 proteins that retain the desired biological activity (e.g., the ability to bind elk or hek) are also within the scope of the present invention. LERK-5 variants may be obtained by mutations of nucleotide sequences coding for native LERK-5 polypeptides. A LERK-5 variant, as referred to herein, is a polypeptide substantially homologous to a native LERK-5, but which has an amino acid sequence different from that of a native LERK-5 due to one or more deletions, insertions or substitutions.
A variant nucleotide or amino acid sequence preferably is at least 80% identical to a native LERK-5 sequence, most preferably at least 90% identical. In one embodiment of the present invention, a LERK-5 protein comprises an extracellular domain, a transmembrane region, and a cytoplasmic domain, wherein the amino acid acid sequence of said LERK-5 protein is at least 90% identical to the sequence presented as amino acids 1 to 308 of SEQ ID NO:2. In another embodiment, a soluble LERK-5 polypeptide capable of binding elk and hek comprises an amino acid sequence that is at least 90% identical to the sequence presented as amino acids 1 to 199 of SEQ ID NO:2.
The percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
Alterations of the native sequence may be accomplished by any of a number of known techniques. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, Jan. 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated by reference herein.
Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
LERK-5 also may be modified to create LERK-5 derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of LERK-5 may be prepared by linking the chemical moieties to functional groups on LERK-5 amino acid side chains or at the N-terminus or C-terminus of a LERK-5 polypeptide or the extracellular domain thereof. Other derivatives of LERK-5 within the scope of this invention include covalent or aggregative conjugates of LERK-5 or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugate may comprise a signal or leader polypeptide sequence (e.g. the xcex1-factor leader of Saccharomyces) at the N-terminus of a LERK-5 polypeptide. The signal or leader peptide co-translationally or post-translationally directs transfer of the conjugate from its site of synthesis to a site inside or outside of the cell membrane or cell wall.
LERK-5 polypeptide fusions can comprise peptides added to facilitate purification and identification of LERK-5. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 (hereby incorporated by reference) and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG(copyright) peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:3), which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody enabling rapid assay and facile purification of expressed recombinant protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing.
The present invention further includes LERK-5 polypeptides with or without associated native-pattern glycosylation. LERK-5 expressed in yeast or mammalian expression systems (e.g., COS-7 cells) may be similar to or significantly different from a native LERK-5 polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of LERK-5 polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules.
N-glycosylation sites in the LERK-5 extracellular domain can be modified to preclude glycosylation. Such sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. The human LERK-5 protein comprises two such triplets, at amino acids 11-13 and 114-116 of SEQ ID NO:2. Appropriate modifications to the nucleotide sequence encoding this triplet will result in substitutions, additions or deletions that prevent attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Pat. No. 5,071,972 and EP 276,846, hereby incorporated by reference.
In another example, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon renaturation. Other variants are prepared by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Argxe2x80x94Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lysxe2x80x94Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lysxe2x80x94Lys represents a conservative and preferred approach to inactivating KEX2 sites. Human LERK-5 contains four KEX2 protease processing sites, at amino acids 228-229, 229-230, 232-233, and 251-252 of SEQ ID NO:2.
Naturally occurring LERK-5 variants are also encompassed by the present invention. Examples of such variants are proteins that result from alternative mRNA splicing events or from proteolytic cleavage of the LERK-5 protein, wherein the elk-binding or hek-binding property is retained. Alternative splicing of mRNA may yield a truncated but biologically active LERK-5 protein, such as a naturally occurring soluble form of the protein, for example. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the LERK-5 protein (generally from 1-5 terminal amino acids).
Variants possessing the requisite ability to bind elk or hek may be identified by any suitable assay. Biological activity of a LERK-5 variant may be determined, for example, by analyzing the variant""s ability to compete with LERK-5 for binding to elk or hek (i.e., in competition binding assays).
Competition binding assays can be performed using standard techniques. For example, radiolabeled LERK-5 can be used to compete with a LERK-5 variant to assay for binding to cell surface-bound elk or hek. Qualitative results can be obtained by competitive autoradiographic plate binding assays, or Scatchard plots may be utilized to generate quantitative results. Instead of intact cells, one could substitute soluble elk or hek bound to a solid phase (e.g., a soluble elk/Fc or hek/Fc fusion protein bound to a solid phase containing Protein A or Protein G) Another type of competitive binding assay utilizes radiolabeled soluble elk or hek, and intact cells expressing LERK-5.
The LERK-5 of the present invention also can be used in a binding assay to detect cells expressing elk or hek. For example, LERK-5 or the extracellular domain thereof can be conjugated to a detectable moiety such as a radionuclide, an enzyme that can catalyze a colorometric or fluorometric reaction, biotin or avidin. Cells to be tested for elk or hek expression are contacted with the labeled LERK-5. After incubation, unbound labeled LERK-5 is separated from the cells, and binding is measured using the detectable moiety.
One aspect of the present invention involves the use of LERK-5 to bind elk or hek proteins. For example, LERK-5 may be employed as a reagent in protein purification procedures. LERK-5 or LERK-5/Fc fusion proteins are attached to a solid support material by conventional techniques and used to purify elk or hek by affinity chromatography.
The LERK-5 proteins disclosed herein also may be employed to measure the biological activity of elk or hek proteins in terms of their binding affinity for LERK-5. As one example, LERK-5 may be used in determining whether biological activity is retained after modification of an elk or hek protein (e.g., chemical modification, truncation, mutation, etc.). The biological activity of an elk or hek protein thus can be ascertained before it is used in a research study, for example.
LERK-5 proteins find use as reagents that may be employed by those conducting xe2x80x9cquality assurancexe2x80x9d studies, e.g., to monitor shelf life and stability of elk protein under different conditions. To illustrate, LERK-5 may be employed in a binding affinity study to measure the biological activity of an elk protein that has been stored at different temperatures, or produced in different cell types. The binding affinity of the modified elk protein for LERK-5 is compared to that of an unmodified elk protein to detect any adverse impact of the modifications on biological activity of elk. Likewise, the biological activity of a hek protein can be assessed using LERK-5.
LERK-5 polypeptides also find use as carriers for delivering agents attached thereto to cells bearing the elk or hek cell surface receptor. Expression of hek antigen has been reported for certain leukemic cell lines, including the human T-cell leukemia cell line designated JM and the human pre-B cell leukemia cell line designated LK63 (Boyd et al., J. Biol. Chem. 267:3262, 1992, and Wicks et al., Proc. Natl. Acad. Sci. USA, 89:1611, 1992). LERK-5 proteins thus can be used to deliver diagnostic or therapeutic agents to these cells (or to other cell types found to express hek on the cell surface) in in vitro or in vivo procedures.
One example of such use is to expose a hek+ leukemic cell line to a therapeutic agent/LERK-5 conjugate to assess whether the agent exhibits cytotoxicity toward the leukemia cells. A number of different therapeutic agents attached to LERK-5 may be included in an assay to detect and compare the cytotoxic effect of the agents on the leukemia cells. LERK-5/diagnostic agent conjugates may be employed to detect the presence of hek+ cells in vitro or in vivo.
Diagnostic and therapeutic agents that may be attached to a LERK-5 polypeptide include, but are not limited to, drugs, toxins, radionuclides, chromophores, enzymes that catalyze a calorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application. Examples of drugs include those used in treating various forms of cancer, e.g., nitrogen mustards such as L-phenylalanine nitrogen mustard or cyclophosphamide, intercalating agents such as cis-diaminodichloroplatinum, antimetabolites such as 5-fluorouracil, vinca alkaloids such as vincristine, and antibiotics such as bleomycin, doxorubicin, daunorubicin, and derivatives thereof. Among the toxins are ricin, abrin, diptheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, 123I, 131I, 99mTc, 111In, and 76Br. Radionuclides suitable for therapeutic use include, but are not limited to, 131I, 211At, 77Br, 186Re, 188Re, 212Pb, 212Bi, 109Pd, 64Cu, and 67Cu.
Such agents may be attached to the LERK-5 by any suitable conventional procedure. LERK-5, being a protein, comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the protein or agent may be derivatized to generate or attach a desired reactive functional group. The derivatization may involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to proteins (Pierce Chemical Company, Rockford, Ill.). A number of techniques for radiolabeling proteins are known. Radionuclide metals may be attached to LERK-5 by using a suitable bifunctional chelating agent, for example.
Conjugates comprising LERK-5 and a suitable diagnostic or thrapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application.
Another use of the LERK-5 of the present invention is as a research tool for studying the role that LERK-5, in conjunction with elk or hek, may play in growth or differentiation of cells bearing the elk or hek receptor. The LERK-5 polypeptides of the present invention also may be employed in in vitro assays for detection of elk or LERK-5 or the interactions thereof. Likewise, LERK-5 finds use in assays for hek or the interaction of LERK-5 with hek.
As discussed above, when various rat tissues were analyzed for elk mRNA, transcripts were detected only in brain and testis (Lhotak et al., supra). Binding of LERK-5 to elk on neural tissue is believed to exert a neuroprotective or neurotrophic effect.
LERK-5 finds use as a tissue culture reagent. A LERK-5 protein can be added to neurons cultured in vitro to enhance the viability or prolong the lifespan of the cultured neurons, thus facilitating research studies of neural tissue.
One embodiment of the present invention is directed to a method of treating disorders of neural tissue, involving contacting the neural tissue with LERK-5. Such disorders include injury or neurologic diseases, either chronic or acute. A LERK-5 protein may be administered to a mammal to treat such an injury or disease. In one embodiment of the invention, LERK-5 is employed in treating neurodegenerative conditions characterized or mediated, at least in part, by the mechanism of neural death known as excitotoxicity. In addition, LERK-5 may be administered to a mammal to exert a trophic effect on neural tissue. In a patient suffering loss of or damage to neurons due to injury or disease, LERK-5 may enhance the viability of those neurons that have survived.
The present invention provides pharmaceutical compositions comprising an effective amount of a purified LERK-5 polypeptide and a suitable diluent, excipient, or carrier. Such carriers will be nontoxic to patients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining a mammalian LERK-5 polypeptide or derivative thereof with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) peptides, proteins, amino acids, carbohydrates including glucose, sucrose, or dextrans, chelating agents such as EDTA, glutathione, or other stabilizers and excipients. Neutral buffered saline is one appropriate diluent.
For therapeutic use, the compositions are administered in a manner and dosage appropriate to the indication and the patient. As will be understood by one skilled in the pertinent field, a therapeutically effective dosage will vary according to such factors as the nature and severity of the condition to be treated, and the age, size, and condition of the patient. Administration may be by any suitable route, including but not limited to continuous infusion, local infusion during surgery, intraventricular infusion (which may involve use of an intraventricular catheter), sustained release from implants (gels, membranes, and the like), or injection (e.g., injection at the site of an injury or injection into the central nervous system).
The compositions of the present invention may contain a LERK-5 protein in any form described above, including variants, derivatives, and biologically active fragments thereof. In one embodiment of the invention the composition comprises a soluble human LERK-5 protein. Such protein may comprise the extracellular domain of human LERK-5 fused to an Fc polypeptide, as described above.
Oligomeric Forms of LERK-5
Encompassed by the present invention are LERK-5 polypeptides in the form of oligomers, such as dimers or trimers. Such oligomers may be naturally occurring or produced by recombinant DNA technology. Oligomers may comprise LERK-5 polypeptides (preferably the extracellular domain or a fragment thereof) linked by disulfide bonds or expressed as a fusion protein with or without spacer peptide linkers. Oligomers may be formed by disulfide bonds between cysteine residues on different LERK-5 polypeptides, for example.
LERK-5 oligomers may be prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al., (PNAS USA 88:10535, 1991) and Byrn et al., (Nature 344:677, 1990), hereby incorporated by reference. In one embodiment of the invention, a LERK-5 dimer is created by fusing LERK-5 to the Fc region of an antibody (IgG1). The Fc polypeptide preferably is fused to the C-terminus of a soluble LERK-5 (comprising only the extracellular domain). A gene fusion encoding the LERK-5/Fc fusion protein is inserted into an appropriate expression vector. The LERK-5/Fc fusion protein is expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between Fc polypeptides, yielding divalent LERK-5. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a LERK-5 oligomer with as many as four LERK-5 extracellular regions.
The term xe2x80x9cFc polypeptidexe2x80x9d as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated froms of such polypeptides containing the hinge region that promotes dimerization are also included. One suitable Fc polypeptide, described in PCT application WO 93/10151, is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus. A mutein of this Fc polypeptide is described in example 3 below. The mutein exhibits reduced affinity for Fc receptors.
Alternatively, one can join LERK-5 polypeptides (preferably two soluble LERK-5 polypeptides) via a peptide linker. Peptide linkers suitable for joining polypeptides are known, and may be employed by conventional techniques. Fusion proteins comprising LERK-5 polypeptides joined by peptide linkers may be produced by recombinant DNA technology, for example.
The present invention provides oligomers of LERK-5 extracellular domains or fragments thereof, linked by disulfide interactions, or expressed as fusion polymers with or without spacer amino acid linking groups. For example, a dimer of the LERK-5 extracellular domain can be linked by an IgG Fc region linking group.
Expression Systems
The present invention provides recombinant expression vectors for expression of LERK-5, and host cells transformed with the expression vectors. Any suitable expression system may be employed. The vectors include a LERK-5 DNA sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the LERK-5 DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a LERK-5 DNA sequence if the promoter nucleotide sequence controls the transcription of the LERK-5 DNA sequence. The ability to replicate in the desired host cells, usually conferred by an origin of replication, and a selection gene by which transformants are identified, may additionally be incorporated into the expression vector.
In addition, sequences encoding appropriate signal peptides that are not native to the LERK-5 gene can be incorporated into expression vectors. For example, a DNA sequence for a signal peptide (secretory leader) may be fused in frame to the LERK-5 sequence so that the LERK-5 is initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells enhances extracellular secretion of the LERK-5 polypeptide. The signal peptide is cleaved from the LERK-5 polypeptide upon secretion of LERK-5 from the cell.
Suitable host cells for expression of LERK-5 polypeptides include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-free translation systems could also be employed to produce LERK-5 polypeptides using RNAs derived from DNA constructs disclosed herein.
Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a LERK-5 polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant LERK-5 polypeptide.
Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. An appropriate promoter and a LERK-5 DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include xcex2-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage xcexPL promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the xcexPL promoter include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).
LERK-5 alternatively may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2xcexc yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657. Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.
The yeast xcex1-factor leader sequence may be employed to direct secretion of the LERK-5 polypeptide. The xcex1-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3xe2x80x2 end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp+transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 xcexcg/ml adenine and 20 xcexcg/ml uracil.
Yeast host cells transformed by vectors containing ADH2 promoter sequence may be grown for inducing expression in a xe2x80x9crichxe2x80x9d medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 xcexcg/ml adenine and 80 xcexcg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
Mammalian or insect host cell culture systems could also be employed to express recombinant LERK-5 polypeptides. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).
Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
Exemplary expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566. Other suitable vectors may be derived from retroviruses.
In place of the native signal sequence, a heterologous signal sequence may be added, such as the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846.
LERK-5 Protein
The present invention provides purified LERK-5 protein, which may be produced by recombinant expression systems as described above or purified from naturally occurring cells. Advantageously, the LERK-5 is purified such that no protein bands corresponding to other proteins are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be appreciated by one skilled in the pertinent field that multiple bands corresponding to LERK-5 protein may be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like, as discussed above. The LERK-5 protein is considered to be purified as long as no bands corresponding to different (non-LERK-5) proteins are visualized. The LERK-5 most preferably is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-PAGE.
One process for producing the LERK-5 protein comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes LERK-5 under conditions such that LERK-5 is expressed. The LERK-5 protein is then recovered from culture medium or cell extracts, depending upon the expression system employed. As the skilled artisan will recognize, procedures for purifying the recombinant LERK-5 will vary according to such factors as the type of host cells employed and whether or not the LERK-5 is secreted into the culture medium.
For example, when expression systems that secrete the recombinant protein are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel having pendant methyl or other aliphatic groups) can be employed to further purify LERK-5. Some or all of the foregoing purification steps, in various combinations, can be employed to provide a substantially homogeneous recombinant protein.
It is also possible to utilize an affinity column comprising the ligand binding domain of elk or hek to affinity-purify expressed LERK-5 polypeptides. LERK-5 polypeptides can be recovered from an affinity column in a high salt elution buffer and then dialyzed into a lower salt buffer for use. Alternatively, an immunoaffinity column may comprise an antibody that binds LERK-5. Soluble LERK-5/Fc fusion proteins may be purified using a chromatography matrix having Protein A or Protein G attached thereto.
Recombinant protein produced in bacterial culture is usually isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
In transformed yeast host cells, LERK-5 is preferably expressed as a secreted polypeptide to simplify purification. The secreted recombinant polypeptide can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal et al. describe a procedure that includes two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.
Nucleic Acids
The present invention further provides LERK-5 nucleotide sequences. Such nucleotide sequences include, but are not limited to, the LERK-5 DNA disclosed herein, in both single-stranded and double-stranded form, as well as the RNA complement thereof. LERK-5 DNA of the present invention includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genonic DNA may be isolated by conventional techniques using the cDNA isolated in example 1, or a suitable fragment thereof, as a probe.
Examples of LERK-5 DNAs of the present invention include, but are not limited to, DNA comprising a nucleotide sequence selected from the group consisting of nucleotides 1 to 1002 of SEQ ID NO:1 (encoding the full length LERK-5 of SEQ ID NO:2); nucleotides 76 to 1002 of SEQ ID NO:1 (encoding a full length mature LERK-5); nucleotides 1 to 672 of SEQ ID NO:1 (encoding the signal peptide and extracellular domain); and nucleotides 76 to 672 of SEQ ID NO:1 (encoding the extracellular domain). Due to the known degeneracy of the genetic code, more than one codon can encode the same amino acid. Thus, a DNA sequence may vary from those described above, yet encode a polypeptide having the same amino acid sequence. Such variant DNA sequences may result from silent mutations, e.g., that may occur during PCR amplification. Alternatively, such silent mutations may be the product of deliberate mutagenesis of a native sequence. DNA sequences that are degenerate as a result of the genetic code to a LERK-5-encoding DNA sequence disclosed herein are encompassed by the present invention.
Useful fragments of the LERK-5 nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target LERK-5 mRNA (sense) or LERK-5 DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of LERK-5 cDNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).
Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of LERK-5 proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oliginucleotide for the target nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO4-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91104753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
The following examples are provided to illustrate particular embodiments and not to limit the scope of the invention.

Antibodies that bind the cytokine designated LERK-5